Somatostatin (SST) is a cyclic tetradecapeptide found in both the central nervous system and peripheral tissues. It was originally isolated from mammalian hypothalamus and identified as an important inhibitor of growth hormone secretion from the anterior pituitary. Its multiple biological activities include inhibition of the secretion of glucagon and insulin from the pancreas, regulation of most gut hormones and regulation of the release of other neurotransmitters involved in motor activity and cognitive processes throughout the central nervous system (for review see Lamberts, Endocrine Rev., 9: 427, 1988). Additionally, SST and its analogs are potentially useful antiproliferative agents for the treatment of various types of tumors. In its natural form, SST has limited use as a therapeutic agent since it exhibits two undesirable properties: poor bioavailability and short duration of action. For these reasons, great efforts have been made to find SST analogs with superior potency, biostability, duration of action and selectivity.
The diverse physiological effects of SST are induced by five selective and high affinity binding to receptors (designated SST-R1 through SST-R5), that are members of the seven transmembrane segment receptor superfamily (reviewed in Reisine T., Bell G I., Endocrinology Rev., 16: 427-442,1995).
Somatostatin in Cancer and Radiolabelled SST Analog
Because SST receptors are present in high density in many endocrine and non-endocrine tumors, diagnosis and treatment were attempted using radiolabelled SST analogs in cancer patients. Most tumors express multiple SST receptor-subtypes, although the SST-R2 subtype is most predominantly expressed. Radiolabelled receptor-specific compounds can detect primary sites, identify occult metastatic lesions, guide surgical intervention, stage tumors, predict efficacy of certain therapeutic agents or, when labelled with suitable radionuclides, be useful radiotherapeutic agents. The abundance of high affinity SST receptors in various tumors enables the use of radiolabelled SST analogs for in vivo identification, visualization and localization of these tumors (Lamberts et al. N. Engl. J. Med. 334:246 1996).
Scintigraphy using radiolabelled SST analog tracers helps to localize tumors and to evaluate the potential for chronic treatment of patients with inoperable SST receptor-positive tumors.
Recently, a number of 99mTc-labeled bioactive peptides have proven to be useful diagnostic imaging agents. Okarvi S. M. (Nuc. Med. Comm., 20:1093, 1999) reviews the recent developments in 99Tc-labeled peptide-based radiopharmaceuticals. Another application of radiolabelled SST analogs is radio-guided surgery. This technique has been successfully utilized in surgery of medullary thyroid cancer, carcinoids and islet cell tumors.
SST receptor radio-imaging has been recently used successfully (Aparici et al. Eur. J. Nuc. Med. 27:1754, 2000), for detection of cardiac allograft rejection. Infiltrated activated lymphocytes express SST receptors thus SST receptor imaging could be used to target them.
Optical Imaging
With the clinical success of radiopeptides as diagnostics and therapeutics, interest in the optical imaging of tumors using fluorescent-labeled peptides has been increasing. In addition, in vivo optical imaging is used to assess specific molecular targets for gene- and cell-based therapies (Allport et al. Exp. Hematol. 29, 1237-1246, 2001). In optical imaging (known also as photodynamic diagnosis or PDD), the patient is neither exposed to externally administered ionizing radiation (e.g., CT) nor injected with radioactive materials (e.g., nuclear medicine radiopharmaceuticals); thus, avoiding both radiation and radioactivity, the technique is more acceptable to the general public. The use of peptide conjugates for optical imaging, demonstrated by in vivo targeting to the somatostatin receptor in rats bearing receptor-expressing tumors have been reported by Licha et al. (Bioconjugate Chem. 12, 44-50, 2001) and Achilefu et al. (Investigative Radiology 35, 479-485, 2000). WO 00/61194 discloses somatostatin peptide-dye conjugates used as contrast agents for optical diagnostics. WO 03/003806 discloses dye-azide compounds, including somatostatin ligands, for dual phototherapy of tumors and other lesions, while WO 03/004466 discloses dye-sulfenate derivatives and their bioconjugates for the same purposes.
U.S. Pat. No. 5,211,938 discloses a method of detection of malignant and non-malignant lesions by photochemotherapy of protoporphyrin IX precursors. JP 2000-342297 discloses cancer cell diagnosis by comparative genomic hybridization with fluorescent labeled probes.
Most of the present methods relating to cancer screening using fluorescence detection systems require the use of interventional devices such as endoscopes which have the special capability of delivering specified light frequencies to a targeted area within a patient. These endoscopes illuminate the targeted part of the body in which cancer is suspected. The light delivered at a specified frequency illuminates an area which has previously been subjected to some type of fluorescent marker, such as a porphyrin which causes malignant cells to illuminate or fluoresce under observation of light at a specified frequency. In these cases, introduction of an endoscope into the body requires some type of sedation or general or local anesthesia. Once a tumor has been located by use of the interventional device, depending upon the type of tumor, photochemotherapy or other treatment means can be used. However, prior to actual treatment, there must be a confirmed test of cancer. Accordingly, the tumor still needs to be sampled by an appropriate biopsy method that also require some type of sedation or anesthesia. Thus, traditional methods of confirming a malignancy may require at least two interventional surgical procedures. U.S. Pat. No. 6,316,215 discloses methods of cancer screening utilizing fluorescence detection techniques and selectable imager charge integration periods. The methods are claimed to allow a safe, reliable, inexpensive and minimally invasive diagnosis.
Photodynamic Therapy
An additional application for optically active receptor-targeted peptide conjugates is in photodynamic therapy (PDT). PDT is a medical technique used mainly for the ablation of cancer. In PDT, a patient is administered a PDT drug, and after the drug has accumulated in the tumor, the tumor is irradiated with light of a specific wavelength using a special light delivery system. Certain photo active molecules (most of them related to porphyrins), when irradiated with light of the proper wavelength in the presence of oxygen, generate reactive oxygen species (ROS) within the cancerous cell which kills the cell, and are therefore useful therapeutically in cancer. In the treatment of cancer, PDT combines light and endogenous oxygen with a photosensitizer localized in or around the tumor. Irradiation of the sensitizer produces a cascade of biochemical events that inactivate cancer cells either directly through attack at specific cellular sites or indirectly through the induction of vascular damage to blood vessels feeding the tumor.
Since many PDT drugs fluoresce at another, distinct wavelength, the same compounds can be used to image cancer using a special spectral imaging device. Spectral imaging can be used to locate a tumor for surgical resection, to locate residual tumor for resection control and to guide subsequent PDT. The therapeutic efficacy of PDT can be monitored in real time by fluorescence imaging during PDT. All of the above procedures are potentially achievable in the operating ward, endoscopically and externally.
An increasing number of regulatory-approved PDT procedures are being put into routine clinical practice. PDT has regulatory approval in the USA, Canada, The Netherlands, France, Germany, and Japan for cancers of the lung, digestive tract, and genitourinary tract. Photofrin® is also being evaluated as a protocol for treating cancers of the head and neck region and for treating pancreatic cancer as well as a possible therapy against Kaposi's sarcoma and cancers of the brain, breast (both primary and metastatic), skin, and abdomen. Although Photofri®n has been shown to be effective against a number of malignancies, it is not the “ideal” photosensitizer. Novel water-soluble porphyrins having improved properties were recently suggested. (Hilmey et al J. Med. Chem. 45, 449-461, 2002).
To date, PDT applications have employed non-receptor-targeted molecules that are taken up in cancer. The mechanisms by which cancer cells take up non-targeted PDT drugs are not well understood, and target to non-target ratios of PDT drug concentrations of only about 2-5 are obtained. There are few reports in the literature of PDT drug receptor targeted biomolecule conjugates demonstrating in vitro efficacy. De Luca et al. (J. Pep. Sci. 7, 386-394, 2001), described synthesis and characterization of a porphyrin-CCK8 conjugate in which the porphyrin is used as an indium chelator, but PDT is not mentioned. Del Govematore et al. (Cancer Res. 60, 4200-4205, 2000), demonstrated in vivo use of a chlorin-antibody construct for targeted PDT of colorectal cancer, but none of the background art publications disclose peptide-receptor-targeted in vivo PDT.
U.S. Pat. Nos. 5,308,608 and 5,149,708 disclose specific types of porphyrin compounds which may be used for detection, photosensitization, or the destruction of a targeted biological material when the targeted tissue is contacted with the specified porphyrin, and irradiated with light that excites the compound. WO 01/15694 and WO 00/41727 disclose methods and compounds for PDT of a target tissue using a light source that preferably transmits light to a treatment site transcutaneously. U.S. Pat. No. 6,054,449 discloses compounds and methods for PDT of intimal hyperplasia and other diseases. U.S. Pat. No. 6,333,319 discloses use of bacteriochlorophyll derivatives in PDT methods, while U.S. Pat. No. 6,147,195, U.S. Pat. No. 5,726,169, U.S. Pat. No. 5,955,585, and U.S. Pat. No. 5,650,292 disclose conjugates of chlorophyll and bacteriochlorophyll derivatives with peptides used as photosensitizers in photodynamic therapy and in diagnostics of tumors. U.S. Pat. No. 6,217,848 discloses cyanine and indocyanine dye-peptide, conjugates for diagnostic imaging and therapy, and specifically discloses somatostatin analogs useful for laser assisted guided surgery for the detection of small micrometastases tumors upon laparoscopy.
Improved Peptide Analogs
As a result of major advances in organic chemistry and in molecular biology, many bioactive peptides can now be prepared in quantities sufficient for pharmacological and clinical use. Thus in the last few years new methods have been established for the treatment and diagnosis of illnesses in which peptides have been implicated. However, the use of peptides as therapeutic and diagnostic agents is limited by the following factors: a) tissue penetration; b) low metabolic stability towards proteolysis in the gastrointestinal tract and in serum; c) poor absorption after oral ingestion, in particular due to their relatively high molecular mass or the lack of specific transport systems or both; d) rapid excretion through the liver and kidneys; and e) undesired side effects in non-target organ systems, since peptide receptors can be widely distributed in an organism.
It would be desirable to achieve peptide analogs with greater specificity thereby achieving enhanced clinical selectivity. It would be most beneficial to produce conformationally constrained peptide analogs overcoming the drawbacks of the native peptide molecules, thereby providing improved therapeutic properties.
A novel conceptual approach to the conformational constraint of peptides was introduced by Gilon et al. (Biopolymers 31:745, 1991) who proposed backbone-to-backbone cyclization of peptides. The advantages of this strategy include the ability to effect cyclization via the carbons or nitrogens of the peptide backbone without interfering with side chains that may be crucial for interaction with the specific receptor of a given peptide. Further disclosures by Gilon and coworkers (WO 95/33765, WO 97/09344, U.S. Pat. Nos 5,723,575, 5,811,392, 5,883,293 and 6,265,375), provided methods for producing building units required in the synthesis of backbone cyclized peptide analogs. The successful use of these methods to produce backbone cyclized peptide analogs of bradykinin (U.S. Pat. No. 5,874,529), and somatostatin (WO 98/04583, WO 99/65508, WO 99/65508, U.S. Pat. Nos. 5,770,687, 6,051,554, and 6,355,613) was also disclosed. WO 02/062819 discloses backbone cyclized radiolabelled SST analogs for radioimaging and therapy. All of these methods are incorporated herein in their entirety, by reference.
There remains a need for synthetic SST analogs having increased in vivo stability, to be used diagnostically and therapeutically, as agents labeled with photo-active moiety, for optical imaging in vivo, in vitro and ex vivo, and for therapeutic agents using photodynamic therapy. It would be desirable to achieve peptide analogs with greater specificity to receptor subtypes thereby achieving enhanced diagnostic selectivity to elucidate the specific SST receptor profile in each individual for planning further therapy and/or surgery. Backbone cyclized SST analogs that specifically fulfill these needs are provided by this invention. None of the background art teaches or suggests the photo-active labeled backbone cyclized somatostatin analogs disclosed herein having improved diagnostic and therapeutic activity and selectivity.